The invention relates to catalytic antibody having aldolase activity. More particularly the invention relates to catalytic antibodies having aldolase activity and which are generated by an immunoconjugate having a xcex2-diketone hapten having suicide substrate activity with respect to such catalytic antibodies.
The aldol addition reaction is a reversible reaction involving the combination of two reactant molecules and the formation of a product having a new carbon-carbon bond. Each of the reactants contains a carbonyl group, i.e., either an aldehyde or ketone. During the reaction, one of the reactants loses a proton from the carbon atom next to its carbonyl group, thereby becoming nucleophilic. The nucleophilic carbon of the first reactant then attacks the carbonyl group of the second reactant. The reverse of this condensation reaction can also occur and entails the cleavage of a carbon-carbon bond and the dissociation of a molecule into two components. The aldol addition reaction is important in the glycolytic pathway and is catalyzed by aldolase enzymes. The aldol addition reaction is also fundamental to organic chemistry for the formation and dissociation of carbon-carbon bonds. In organic chemistry, the reaction may be catalyzed by base.
Two mechanistic classes of aldolase enzymes have evolved, viz., Class I and Class II aldolases. (W. J. Rutter, Fed. Proc. Amer. Soc. Exp. Biol. (1964): vol. 23, p 1248.) Class I aldolases utilize the xcex5-amino group of a Lys in the active site to form a Schiff base with one of the substrates, which activates the substrate as an aldol donor.
The mechanism for class I aldolases is illustrated in FIG. 1. The reaction is bimolecular and proceeds through covalent catalysis through multiple intermediates. An iminium ion or Schiff base forms that acts as an electron sink, which lowers the activation energy (Ea) for proton abstraction from Cxcex1 and subsequent enamine formation. The enamine acts as the carbon nucleophile, or aldol donor, which reacts with an aldehyde electrophile, the aldol acceptor, to form a new Cxe2x80x94C bond. The Schiff base is then hydrolyzed and the product is released. The essence of the mechanism is the formation of the enamine which is the nascent carbon nucleophile.
Class II aldolases are metalloenzymes that facilitate enolate formation by coordination to the substrate""s carbonyl oxygen. Transition state models have also been disclosed for aldol reactions involving metals. (H. E. Zimmerman et al., J. Am. Chem. Soc. (1957): vol. 79, p 1920.) However, the mechanism for Class II aldolases remains to be fully characterized.
A number of enzymes catalyze the aldol condensation. The mechanisms of these enzymes have been well characterized. (C. Y. Lai, et al., Science (1974): vol. 183, p 1204; and A. J. Morris e al., Biochemistry (1994) vol. 33, p 12291.) However, aldolase enzymes accept a relatively limited range of substrates (C. -H. Wong et al., Enzymes in Synthetic Organic Chemistry (Permagon, Oxford, 1994); M. D. Bednarski in Comprehensive Organic Synthesis, B. M. Trost, Ed.(Pergamon, Oxford, 1991), vol 2, pp. 455-473; C. F. Barbas III, et al., J. Am. Chem. Soc. (1990): vol 112, p 2013; H. J. M. Gijsen et al., J. Am. Chem. Soc. (1995): vol. 117, p 2947; C. -H. Wong et al., J. Am. Chem. Soc. (1995): vol. 117, p. 3333; L. Chen, et al., J. Am. Chem. Soc (1992): vol. 114, p 741.) Although natural aldolase enzymes display broad specificity with respect to the aldol acceptor, the aldol donor is usually limited to the natural substrate. The art of organic synthesis would benefit significantly if catalysts having the desired substrate specificity could be produced to order for catalyzing desired aldol addition reactions.
Non-enzymic base catalyzed aldol addition reactions are employed widely in organic chemistry to form new carbonxe2x80x94carbon bonds. Also, a variety of effective reagents have been developed to control the stereochemistry of the aldol. However, these reagents are stoichiometric and require pre-formed enolates and extensive protecting group chemistry. (C. H. Heathcock, Aldrichim. Acta (1990): vol. 23, p 99; C. H. Heathcock, Science (1981): vol. 214, p 395; D. A. Evans, Science (1988): vol. 240, p 420; S. Masamune, et al., Angew. Chem. Int. Ed. Engl. (1985): vol. 24, p 1; D. A. Evans, et al., Top. Stereochem. (1982): vol. 13, p 1; C. H. Heathcocket et al., in Comprehensive Organic Synthesis, B. M. Trost, Ed. (Pergamon, Oxford, 1991), vol. 2, pp. 133-319 (1991); and I. Paterson, Pure and Appl. Chem. (1992): vol. 64, 1821.) Recently catalytic aldol reactions that use pre-formed enolates have been developed, including the Mukaiyama cross-coupling aldol. (S. Kobayashi, et al., Tetrahedron (1993): vol. 49, p 1761; K. Furuta, et al., J. Am. Chem. Soc. (1991): vol. 113, p 1041; T. Bach, Angew. Chem. Int. Ed. Engl. (1994): vol. 33, p 417 and references therein; and E. M. Carreira, et al., J. Am. Chem. Soc. (1995): vol. 117, p 3649.)
For some reactions, the problem of complex intermediates may be solved by using relatively reactive compounds rather than the more usual inert antigens to immunize animals or select antibodies from libraries such that the process of antibody induction involves an actual chemical reaction in the binding site. (C. F. Barbas III, et al., Proc. Natl. Acad. Sci. USA (1991): vol. 88, p 7978 (1991); K. D. Janda et al., Proc. Natl. Acad. Sci. USA (1994): vol. 191, p 2532.) This same reaction then becomes part of the catalytic mechanism when the antibody interacts with a substrate that shares chemical reactivity with the antigen used to induce it.
One of the major goals of organic chemistry is to use the understanding of reaction mechanisms to design new catalysts. This is often not easy because one must address intermediates that are of high energy and complex structure. Antibody catalysts offer one potential solution to this problem in that they can be programmed by the experimenter to interact with the rate limiting transition state of a chemical reaction thereby lowering its energy and increasing the reaction rate. (R. A. Lerner, et al., Science (1991): vol. 252, p 659.) However, even here the ability of the experimenter to program the catalyst is usually limited to the more global aspects of the transition state rather than the detailed reaction mechanism. Thus, while one can deal with high energy charges, stereoelectronic, and geometrical features that appear along the reaction coordinate, the organization of multiple complex reaction intermediates remains difficult.
What is needed is a method for inducing antibodies that use the reaction mechanisms that give aldolases their efficiency but that take advantage of the range of substrates and stereochemical specificities available with antibodies. What is need is a strategy which would amalgamate the best features of the simple chemical and enzymatic approaches to the problem of forming carbon-carbon bonds via the aldol condensation which is, arguably, the most basic Cxe2x80x94C bond forming reaction in chemistry and biology.
The invention is directed to the generation of antibodies that catalyze the aldol reaction. The catalytic antibodies are generated by immunization with a reactive compound that covalently traps a Lysine (Lys) residue in the binding pocket of the antibody by formation of a stable vinylogous amide, i.e., a covalent antibody/hapten complex. The catalytic mechanism for these catalytic antibodies is disclosed to mimic the catalytic mechanism employed by natural class I aldolase enzymes.
The same reaction mechanism employed to form the covalent antibody/hapten complex is also employed to catalyze the aldol reaction. During catalysis, the antibodies use the e-amino group of Lys to form an enamine with ketone substrates and then use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The catalytic antibodies disclosed herein are characterized by their broad substrate specificity and their ability to control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.
More particularly, one aspect of the invention is directed to antibody molecules or molecules containing antibody combining site portions that catalyze an aldol addition reaction between an aliphatic ketone donor and an aldehyde acceptor. These antibodies are characterized by having a lysine with an e-amino group. They are further characterized by being subject to inhibition with the a xcex2-diketone hapten by formation of a complex between the xcex2-diketone hapten and the e-amino group of the lysine of the catalytic antibody. The complex being may be a stable covalent vinylogous amide, a conjugated enamine, or a Schiff base. In a preferred embodiment, the antibody molecules control the diastereofacial selectivity of the aldol addition reaction in both Cram-Felkin and anti-Cram-Felkin directions. Preferred aliphatic ketone donors include compounds represented by the following structures: 
Preferred aldehyde acceptor include compounds represented by the following structures: 
Another aspect of the invention is directed to molecules of claim 1 what are secreted by hybridoma 38C2, having ATCC accession number HB 12005 or by hybridoma 33F12, having ATCC accession number HB 12004.
Another aspect of the invention is directed to cells that when cultured in a medium produce the above indicated monoclonal antibody molecules or molecules containing antibody combining site portions that catalyze an aldol addition reactions. In a preferred embodiment, the cells are to a type that secrete into the culture medium the monoclonal antibody molecules or molecules containing antibody combining site portions. Hybridoma cells are a preferred embodiment, viz., hybridoma cells of hybridoma 38C2, having ATCC accession number HB 12005 and hybridoma cells of hybridoma 33F12, having ATCC accession number HB 12004.
A further aspect of the invention is directed to a method for catalyzing an aldol addition reaction between an aliphatic ketone donor and an aldehyde acceptor. The method begins by admixing a catalytically effective amount of the monoclonal antibody molecules or molecules containing antibody combining site portions with the aliphatic ketone donor and said aldehyde acceptor in an aqueous medium to form a reaction admixture. After the reaction admixture is formed, it is maintained for a period of time sufficient for the antibody molecules or molecules containing antibody combining site portions to catalyze the aldol addition reaction between the aliphatic ketone donor and the aldehyde acceptor. In a preferred mode of the above synthetic method, the antibody molecules or molecules containing antibody combining site portions thereof are secreted by hybridoma 38C2, having ATCC accession number HB 12005 or by hybridoma 33F12, having ATCC accession number HB 12004.
An alternative mode of the invention is directed to a process for carrying out an aldol addition reaction by forming a reaction mixture by admixing an aliphatic ketone donor, an aldehyde acceptor, and a catalytically effective amount of monoclonal antibodies or paratope-containing portions of the monoclonal antibodies in an aqueous medium at a pH value between about 6 and 10. The monoclonal antibodies or paratope-containing portions thereof are of a type which include a lysine with an xcex5-amino group which reacts with the aliphatic ketone donor to form an enamine intermediate. After the reaction mixture is formed, it is maintained under biological reaction conditions for a time period sufficient for the enamine intermediate to react with the aldehyde acceptor to form an aldol addition product.
Another aspect of the invention is directed to a method for preparing cells that when cultured in a medium produce antibody molecules or molecules containing antibody combining site portions that catalyze an aldol addition reaction between an aliphatic donor and a aldehyde acceptor. The method starts by immunizing an animal with an immunogen that includes a xcex2-diketone hapten. Then the animal is maintained for a time period sufficient for it to secrete antibodies that immunoreact with the haptenic ligand. Then genes that encode antibody molecules or molecules containing antibody combining site portions are transferred from antibody-producing cells of the maintained, immunized animal into host cells to form hybrid cells. The hybrid host cells contain genes from at least two sources. The formed hybrid cells have two characteristics, viz.,(i) they produce antibody molecules or molecules containing antibody combining site portions from the transferred genes when cultured and (ii) they can be cultured substantially indefinitely. Then, the hybrid cells are cultured in an appropriate culture medium for a time period sufficient for them to produce antibody molecules or molecules containing antibody combining site portions. Next, antibody molecules or molecules containing antibody combining site portions are recovered from the cultured hybrid cells. Then, the obtained antibody molecules or molecules containing antibody combining site portions are screened for catalytic activity directed to the aldol addition reaction. And finally, clones are grown of the identified hybrid cell that produces antibody molecules or molecules containing antibody combining site portions that catalyze the aldol addition reaction between the aliphatic donor and the aldehyde acceptor. Preferred hybrid cells are hybridoma cells.